The pivotal role of Ca2+ in regulating pancreatic acinar cell function under normal and pathophysiological conditions is well established, however, the molecular changes that occur in response to elevated Ca2+ are largely unknown. This proposal addresses a novel mechanism by which cytosolic Ca2+ modulates the trafficking of phospholipids and associated regulatory proteins in the secretory and endocytic pathways in acinar cells. Calcium responsive heat-stable protein (CRHSP-28) is a key regulatory molecule in the secretory pathway of acinar cells. CRHSP-28 is highly modulated by changes in cellular Ca2+ as indicated by its Ca2+-dependent 1) regulation of digestive enzyme secretion, 2) interaction with the vesicletrafficking protein annexin VI, and 3) serine phosphorylation, which triggers the release of CRHSP-28 from a membrane associated complex. The primary objective of this proposal is to test the hypothesis that CRHSP- 28 acts as Ca2+-sensor to promote and stabilize key protein interactions necessary for acinar cell membrane trafficking. In Specific Aim 1 experiments will address the concept that interaction of CRHSP-28 with annexin VI directs CRHSP-28 association with endosomes that are necessary to support secretory function. Site specific mutants targeting the annexin VI binding domain will be expressed in acini and effects on zymogen secretion and membrane trafficking determined. Specific Aim 2 will utilize CRHSP-28 mutants that alter the major CRHSP-28 phosphorylation site, serine 136, to test the theory that phosphorylation inhibits CRHSP-28 function by displacing it from a membrane-bound state. Specific Aim 3 will address the hypothesis that CRHSP-28 regulates an apical membrane trafficking pathway that is distinct from zymogen granules and acts to shuttle important regulatory molecules to the apical membrane. Elucidation of the molecular mechanism by which CRHSP-28 modulates exocrine function should provide valuable insight into the.Ca2+- dependent nature of the secretory pathway in acinar cells, which is essential for the development of therapeutic strategies aimed at the treatment of exocrine pancreatic disease. This proposal is aimed at understanding the biochemical mechanism by which changes in cell Ca2+ regulate pancreatic function in normal and pathological states. As such, these studies will help to identify potential targets for drug discovery and therapeutic strategies ajmed at treating pancreatic disease.